Angel Peggi M, Orlando Ron
Complex Carbohydrate Research Center, The Departments of Biochemistry & Molecular Biology and Chemistry, University of Georgia, 315 Riverbend Road, Athens, GA 30302-4712, USA.
Rapid Commun Mass Spectrom. 2007;21(10):1623-34. doi: 10.1002/rcm.2990.
A method was developed that uses urea to both solublize and isotopically label biological samples for comparative proteomics. This approach uses either light or heavy urea ((12)CH(4)(14)N(2)O or (13)CH(4)(15)N(2)O, respectively) at a concentration of 8 M and a pH of 7 to dissolve the samples prior to digestion. After the sample is digested using standard proteomic protocols and dried, isotopic labeling is completed by resuspending the sample in a solution of 8 M urea at a pH of 8.5, using the same isotopic species of urea as used for digestion and incubating for 4 h at 80 degrees C. Under these conditions, carbamylation occurs only on the primary amines of the peptides. The effects of complete carbamylation on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) (collision-induced dissociation (CID)) were examined. Peptides that had a C-terminal carbamylated lysine residue were found to have a reduced intensity when viewed by MALDI-TOFMS. CID of a tryptic peptide that was carbamylated on both the N-terminus and the C-terminus was found to have a more uniform distribution of b- and y-ions, as well as prominent ions from loss of water. Reversed-phase chromatography coupled to ESI-MS/MS was used to identify and quantify the isotopically labeled standard proteins, bovine serum albumin (BSA), bovine transferrin, and bovine alpha-casein. Quantitative error between theoretical and observed data ranged from 1.7-10.0%. Relative standard deviations for protein quantitation ranged from 5.2-27.8% over a dynamic range from 0.1-10 (L/H). The development of a method utilizing urea-assisted carbamylation of lysines and N-termini to globally labeled samples for comparative proteomics may prove useful for samples that require a strong chaotrope prior to proteolysis.
开发了一种方法，该方法使用尿素溶解生物样品并对其进行同位素标记，用于比较蛋白质组学。此方法使用浓度为8 M、pH值为7的轻尿素或重尿素（分别为(12)CH(4)(14)N(2)O或(13)CH(4)(15)N(2)O）在消化前溶解样品。使用标准蛋白质组学方案消化样品并干燥后，通过将样品重悬于pH值为8.5的8 M尿素溶液中，使用与消化时相同的同位素种类的尿素，并在80℃下孵育4小时来完成同位素标记。在这些条件下，氨甲酰化仅发生在肽的伯胺上。研究了完全氨甲酰化对基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）和电喷雾电离串联质谱（ESI-MS/MS）（碰撞诱导解离（CID））的影响。发现具有C端氨甲酰化赖氨酸残基的肽在用MALDI-TOFMS观察时强度降低。发现N端和C端均被氨甲酰化的胰蛋白酶肽的CID具有更均匀分布的b离子和y离子，以及失水产生的突出离子。反相色谱与ESI-MS/MS联用用于鉴定和定量同位素标记的标准蛋白质，牛血清白蛋白（BSA）、牛转铁蛋白和牛α-酪蛋白。理论数据与观察数据之间的定量误差范围为1.7 - 10.0%。在0.1 - 10（L/H）的动态范围内，蛋白质定量的相对标准偏差范围为5.2 - 27.8%。开发一种利用赖氨酸和N端的尿素辅助氨甲酰化对样品进行全局标记以用于比较蛋白质组学的方法，对于在蛋白水解前需要强变性剂的样品可能是有用的。
